Using a highly sensitive pulsed-flow microcalorimeter, we have determined the changes in enthalpy and heat capacity for Cro repressor protein-DNA association reactions. The effect of base pair substitutions in the operator region of synthetic oligomeric (21 bp) DNAs upon the thermodynamics of reaction with a mutant (Val 27) and wild type Cro was investigated. (1) The deltaH accompanying formation of the specific Cro repressor- operator OR3 DNA complex is endothermic below 18 degrees C and exothermic above 18 degrees C. At 15 degrees C, deltaC(p)=-360 cal mol(-1)deg(-1). (2) The deltaH upon formation of the nonspecific DNA complex is about +4 kcal mol(-1) and deltaC(p) is zero. (3)deltaC(p) values for the formation of specific complexes with intermediate affinities (in binding of DNA and protein mutants) fall in between. There is a strong correlation between deltaG degrees for specific binding (=deltaG(o) specific -delatG(o)nonspecific) and heat capacity change given by delta deltaG(o)(cal mol-1) = 18 (deg) x delta deltaC(p) (cal mol-1deg-1). No correlation is observed between other thermodynamic parameters. Examination of the data for the reactions of Cro with base pair substitution mutants suggests that the differences in the measured values of the thermodynamic parameters derive from perturbations of specific hydrogen bonds and van der Waals' interactions within the complex rather than from differences in general interactions with the solvent environment.